Polyketides as Secondary Metabolites from the Genus Aspergillus

Polyketides are an important class of structurally diverse natural products derived from a precursor molecule consisting of a chain of alternating ketone and methylene groups. These compounds have attracted the worldwide attention of pharmaceutical researchers since they are endowed with a wide array of biological properties. As one of the most common filamentous fungi in nature, Aspergillus spp. is well known as an excellent producer of polyketide compounds with therapeutic potential. By extensive literature search and data analysis, this review comprehensively summarizes Aspergillus-derived polyketides for the first time, regarding their occurrences, chemical structures and bioactivities as well as biosynthetic logics.

As one of the ubiquitous fungi in nature, the genus Aspergillus has recently received much more attention owing to its great biosynthetic potential of secondary metabolites (SMs) with nutritional, agrochemical and medicinal applications [8]. By the end of 2022, over 3100 Aspergillus-derived SMs had been isolated and collected in the Dictionary of Natural Products (DNP) database [9]. Among these substances, as many as 343 polyketide derivatives (1-343) had been discovered and characterized from Aspergillus strains. To enrich our knowledge of these molecules and explore their therapeutic potentials, all aspects are well organized and comprehensively summarized in this review, including their biological sources, structural features, biological properties as well as biosynthetic logic.

Furans and Furanones
Furans and furanones are the most polyketides produced by Aspergillus spp. and display a broad spectrum of biological properties [23]. Structurally, these substances are classified into two major types, including furans and benzofurans ( Figure 3) and furanones and benzofuranones (Figures 4-6).

Furans and Furanones
Furans and furanones are the most polyketides produced by Aspergillus spp. and display a broad spectrum of biological properties [23]. Structurally, these substances are classified into two major types, including furans and benzofurans (

Furans and Benzofurans
Chemical investigation of one A. niger strain from the Caribbean sponge Hyrtios pro-

Furans and Benzofurans
Chemical investigation of one A. niger strain from the Caribbean sponge Hyrtios proteus led to the discovery of a new furan with a unique carbon skeleton, asperic acid (32) [24], which was later reisolated from the strain A. phoenicis collected in Saskatchewan (Canada) and exhibited potent cytotoxic activity toward the murine lymphocytic leukemia P388 with an ED 50 value of 0.18 mug/mL and a variety of human cancer cell lines (pancreas, breast, CNS, lung, colon, and prostate) with GI 50 values ranged from 1.7 to 2.0 µg/mL [25]. Asperfuranone (33) was a novel polyketide consisting of a conjugated alkene chain and a furan subunit produced by A. nidulans by replacing the promoter of the transcription activator with the inducible alcA promoter [26]. A gene cluster containing two fungal PKSs (AN1036.3 and AN1034.3) for the biosynthesis of 33 was first characterized (Scheme 2), and its mechanism of action (MOA) showed that this compound exerted an inhibitory effect on A549 cells via blocking cell cycle progression and inducing apoptosis [27]. Two prenylated benzaldehyde derivatives (34 and 35) were characterized from the marinederived fungus A. glaucus HB1-19 and showed strong radical-scavenging activity [28]. A new benzofuran polyketide (36) was produced by soil fungus A. terreus X3 but displayed no antimicrobial effect [29]. Flufuran (37) was a typical furan polyketide discovered from A. flavus 9643 and shown to inhibit Phytophthora cinnamomi at 0.2 mg/mL [30,31]. 3,7-Dihydroxy-1,9-dimethyldibenzo-furan (38) originally obtained from a mycobiont of the lichen Lecanora cinereocarnea was found to be produced by an endozoic fungus A. sydowii SCSIO 41301 from marine sponge Phakellia fusca [32,33]. Asperochratide H (39) was a new cytotoxic C 9 polyketide produced by the deep-sea-derived fungus A. ochraceus, and its putative biosynthetic route was proposed in Scheme 3 [34]. Asperpentenone A (40) possesses a rare cyclopentenone-tetrahydrofuran moiety from strain Aspergillus sp. SCSIO 41024 [35]. Asticolorins A-C (41-43) are toxic metabolites manufactured by strain A. versicolor MRC 638 and were characterized by the novel way in which a mevalonatederived 3,3-dimethylallyl group was used to link two dibenzofuran moieties [36,37].

Furans and Benzofurans
Chemical investigation of one A. niger strain from the Caribbean sponge Hyrtios proteus led to the discovery of a new furan with a unique carbon skeleton, asperic acid (32) [24], which was later reisolated from the strain A. phoenicis collected in Saskatchewan (Canada) and exhibited potent cytotoxic activity toward the murine lymphocytic leukemia P388 with an ED50 value of 0.18 mug/mL and a variety of human cancer cell lines (pancreas, breast, CNS, lung, colon, and prostate) with GI50 values ranged from 1.7 to 2.0 μg/mL [25]. Asperfuranone (33) was a novel polyketide consisting of a conjugated alkene chain and a furan subunit produced by A. nidulans by replacing the promoter of the transcription activator with the inducible alcA promoter [26]. A gene cluster containing two fungal PKSs (AN1036.3 and AN1034.3) for the biosynthesis of 33 was first characterized (Scheme 2), and its mechanism of action (MOA) showed that this compound exerted an inhibitory effect on A549 cells via blocking cell cycle progression and inducing apoptosis [27]. Two prenylated benzaldehyde derivatives (34 and 35) were characterized from the marine-derived fungus A. glaucus HB1-19 and showed strong radical-scavenging activity [28]. A new benzofuran polyketide (36) was produced by soil fungus A. terreus X3 but displayed no antimicrobial effect [29]. Flufuran (37) was a typical furan polyketide discovered from A. flavus 9643 and shown to inhibit Phytophthora cinnamomi at 0.2 mg/mL [30,31]. 3,7-Dihydroxy-1,9-dimethyldibenzo-furan (38) originally obtained from a mycobiont of the lichen Lecanora cinereocarnea was found to be produced by an endozoic fungus A. sydowii SCSIO 41301 from marine sponge Phakellia fusca [32,33]. Asperochratide H (39) was a new cytotoxic C9 polyketide produced by the deep-sea-derived fungus A. ochraceus, and its putative biosynthetic route was proposed in Scheme 3 [34]. Asperpentenone A (40) possesses a rare cyclopentenone-tetrahydrofuran moiety from strain Aspergillus sp. SCSIO 41024 [35]. Asticolorins A-C (41-43) are toxic metabolites manufactured by strain A. versicolor MRC 638 and were characterized by the novel way in which a mevalonate-derived 3,3-dimethylallyl group was used to link two dibenzofuran moieties [36,37].
Aspergones A-D (63-66) were detected in the fermentation broth of a marine spongederived strain Aspergillus sp. OUCMDZ-1583 and compounds 63 and 64 showed an inhibitory effect on α-glucosidase with IC 50 values of 2.36 and 1.65 mM, respectively [45]. Dihydropenicillic acid (67) was purified from the endophytic fungus A. flocculus [22] but displayed no antimicrobial or cytotoxic activity [46]. Asperochratide F (68) was another new C 9 polyketide from the deep-sea-derived fungus A. ochraceus and exerted significant cytotoxic effects on the BV-2 cell line [34]. Gregation B (69) was a rare β-furanone derived from A. flavus in food samples by a qualitative analytical method based on the identification of fungal chemical markers by HPLC-MS [30] and exhibited antibacterial activity against E. coli [47]. Avenaciolide (70) produced by strain A. avenaceous G. Smith displayed an inhibitory effect on the transport of glutamate in rat liver mitochondria [48,49]. Citrifurans A−D (71-74) was the first heterodimers of azaphilone and furanone from a symbiotic Aspergillus strain in the intestines of centipedes and displayed moderate inhibitory activities against LPS-induced NO production in RAW 264.7 macrophages [50]. One year later, two additional new β-furanones (75 and 76) were obtained from the same strain, and 76 showed significant NO inhibition with an IC 50 value of 16.0 µM [51].

Naphthalenes
Naphthalenes, a kind of polycyclic aromatic hydrocarbon composed of two benzene rings sharing two adjacent carbon atoms, are toxic to the liver and nervous system and usually cause cataracts and retinal hemorrhage [77,78]. Six naphthalenes 137-142 ( Figure  9) were separated from the marine-derived fungus A. glaucus but showed no cytotoxicity at 100 µ M against the HL-60 and A-549 cell lines [79]. Using heterologous expression in model host A. nidulans RJMP1.49, three analogs neosartoricins B-D (143-145) were biosynthesized and identified [80]. Funalenone (146) was produced by an epigenetic regulator gene-deleted strain A. niger FGSC A1279 and displayed an inhibitory effect on type I collagenase activity at 170 µ M [81]. Two hydroxynaphthalene-2-carboxylate (147,148) were derived from the marine fungus A. terreus SCSIO 41008 and showed weak or no cytotoxic activities toward human glioma U87 cells and glutamate-induced toxicity in HT22 cells [69].

Naphthalenes
Naphthalenes, a kind of polycyclic aromatic hydrocarbon composed of two benzene rings sharing two adjacent carbon atoms, are toxic to the liver and nervous system and usually cause cataracts and retinal hemorrhage [77,78]. Six naphthalenes 137-142 ( Figure 9) were separated from the marine-derived fungus A. glaucus but showed no cytotoxicity at 100 µM against the HL-60 and A-549 cell lines [79]. Using heterologous expression in model host A. nidulans RJMP1.49, three analogs neosartoricins B-D (143)(144)(145) were biosynthesized and identified [80]. Funalenone (146) was produced by an epigenetic regulator gene-deleted strain A. niger FGSC A1279 and displayed an inhibitory effect on type I collagenase activity at 170 µM [81]. Two hydroxynaphthalene-2-carboxylate (147,148) were derived from the marine fungus A. terreus SCSIO 41008 and showed weak or no cytotoxic activities toward human glioma U87 cells and glutamate-induced toxicity in HT22 cells [69].

Polyenes
Polyene polyketides are one kind of important antibiotic which are widely used in the treatment of microbial infections [95]. Structurally, Aspergillus-derived polyenes are linear chain molecules ( Figure 11). Fumagillin (177), discovered from Aspergillus sp. in

Pyrans
Recently pyran derivatives received more and more attention due to their wide biological activities, including antibacterial and antifungal activities, and many of them have

Pyrans
Recently pyran derivatives received more and more attention due to their wide biological activities, including antibacterial and antifungal activities, and many of them have been developed as commercial antimicrobial agents, such as triadimefon, triadimenol, diniconazole, myclobutanil and bitertanol [100,101]. Azaphilones (189-193, Figure 12) are a class of highly oxygenated pyrano-quinone bicyclic chemicals from strain A. niger ATCC 1015 by activation of a silent PKS gene (aza) [102]. Moreover, their biosynthetic pathways were shown to involve the convergent actions of a highly reducing PKS and a non-reducing PKS. Citrinin (194) is a pyran mycotoxin produced by several strains of Aspergillus, Penicillium and Monascus. In addition to toxicity, this compound displayed certain anticancer and neuroprotective effects [103].

Pyranones
Protulactone B (202, Figure 13) was a new α-pyranone polyketide possessing unique ring systems isolated from an EtOAc extract of the marine-derived fungus A. sp. SF-5044 [54]. Chaetoquadrin F (203) produced by strain A. sp. 16-02-1 showed antitumor activity against HeLa cell lines with an inhibitory rate (IR) of 13.5% at 100 μg/mL [43]. In addition to asperochrins A (82), five pyranone derivatives (204-208) were also obtained from strain A. ochraceus MA-15 and compounds 205 and 206 displayed inhibitory activity against aquatic pathogens A. hydrophila, V. anguillarum, and V. harveyi [44]. By the heterologous expression of the avirulence gene ACE1 in A. oryzae M-2-3, two new polyenyl-α-pyranones (209 and 210) were produced and shown to be not responsible for the observed ACE1-mediated avirulence [105]. (+)-Asperlin (211) was discovered from an A. nidulans mutant, which fused the DNA-binding domain of a transcription factor associated with a silent SM gene cluster with the activation domain of a robust SM transcription factor AfoA [106]. Deletion of the epigenetic regulator gene, a histone acetyltransferase in the SAGA/ADA complex, resulted in the production of a novel compound, nigerpyrone (212) in A. niger FGSC A1279 [107]. Moreover, its biosynthetic pathway was disclosed via gene knockout and complementation experiments (Scheme 4). Aspopyrone A (213) was produced by an Okinawan plant-derived fungus, A. sp. TMPU1623 exhibited a strong inhibitory effect on protein tyrosine phosphatase (PTP) 1B with an IC50 value of  (218) were also produced by the marine strain A. sydowii SCSIO 41301 [33], and phomapyrone C (219) together with compounds 40, 176 and 215 was purified from strain SCSIO 41024 [35].

Quinones
Quinones constitute an important class of naturally occurring compounds containing unsaturated cyclic ketone(s) [110]. On the basis of chemical structure, Aspergillus-derived quinones (230-277) could be divided into three types, including anthraquinone, benzoquinone and naphthoquinone, in which the first is the major subgroup [111].

Steroids
Steroids are cyclopentane polyhydrophenanthrenes and play an important role in life activities [125,126]. Ergosterol (278, Figure 17) was isolated and identified from an endophytic strain A. sp. TJ23 and exhibited anticancer activities against cell lines B16, MDA-MB-231, 4Tl, HepG2 and LLC with IC50 values ranging from 5.13 to 12.3 µ M [63]. An ergosterol peroxide (279) and campesterol (280) were obtained from the fermentation culture of an oyster-derived A. flocculus by using modern metabolomics technology [22], and the former displayed an inhibitory effect on the migration of MDA-MB-231 cells at <20 µ M [127,128]. An epoxide steroid (281) was discovered from the deep-sea strain A. sp. SCSIO 41017 was shown to possess moderate activity against cancer cell lines SF-268,

Steroids
Steroids are cyclopentane polyhydrophenanthrenes and play an important role in life activities [125,126]. Ergosterol (278, Figure 17) was isolated and identified from an endophytic strain A. sp. TJ23 and exhibited anticancer activities against cell lines B16, MDA-MB-231, 4Tl, HepG2 and LLC with IC 50 values ranging from 5.13 to 12.3 µM [63]. An ergosterol peroxide (279) and campesterol (280) were obtained from the fermentation culture of an oyster-derived A. flocculus by using modern metabolomics technology [22], and the former displayed an inhibitory effect on the migration of MDA-MB-231 cells at <20 µM [127,128]. An epoxide steroid (281) was discovered from the deep-sea strain A. sp. SCSIO 41017 was shown to possess moderate activity against cancer cell lines SF-268, MCF-7, HepG-2 and A549 with IC 50 values of 13.5-18.0 µM [129].

Steroids
Steroids are cyclopentane polyhydrophenanthrenes and play an important role in life activities [125,126]. Ergosterol (278, Figure 17) was isolated and identified from an endophytic strain A. sp. TJ23 and exhibited anticancer activities against cell lines B16, MDA-MB-231, 4Tl, HepG2 and LLC with IC50 values ranging from 5.13 to 12.3 µ M [63]. An ergosterol peroxide (279) and campesterol (280) were obtained from the fermentation culture of an oyster-derived A. flocculus by using modern metabolomics technology [22], and the former displayed an inhibitory effect on the migration of MDA-MB-231 cells at <20 µ M [127,128]. An epoxide steroid (281) was discovered from the deep-sea strain A. sp. SCSIO 41017 was shown to possess moderate activity against cancer cell lines SF-268, MCF-7, HepG-2 and A549 with IC50 values of 13.5-18.0 µ M [129].

Meroterpenoids
Meroterpenoids as polyketide-terpenoid hybrids are a family of fungal metabolites possessing significant biological activities [130]. However, only a small group of meroterpenoids (282-292, Figure 18) had been isolated and characterized from Aspergillus strains. Terretonin (282), produced by a strain of A. terreus, had a novel, heavily oxidized 25-carbon skeleton and was presumably derived from the degradation of a triterpene precursor [131]. Co-cultivation of a strain of A. fumigatus with the actinomycete Streptomyces rapamycinicus afforded the production of two new prenylated polyketides (283 and 284) Figure 17. Aspergillus-derived steroids (278-281).

Meroterpenoids
Meroterpenoids as polyketide-terpenoid hybrids are a family of fungal metabolites possessing significant biological activities [130]. However, only a small group of meroterpenoids (282-292, Figure 18) had been isolated and characterized from Aspergillus strains. Terretonin (282), produced by a strain of A. terreus, had a novel, heavily oxidized 25-carbon skeleton and was presumably derived from the degradation of a triterpene precursor [131]. Co-cultivation of a strain of A. fumigatus with the actinomycete Streptomyces rapamycinicus afforded the production of two new prenylated polyketides (283 and 284) [132]. Parasiticolide A (285) was the common SM of two strains of A. flavus and A. parasiticus IFO 4082 [30,133]  Additionally, chemical analysis of the liquid cultures of strain TJ23 resulted in the discovery of two novel terpene-polyketide hybrids (287 and 288), of which compound 287 was a potential inhibitor of PBP2a and worked synergistically with the β-lactam antibiotics oxacillin and piperacillin against MRSA [135]. Sphaeropsidin A (289), along with aspergiloid E (290), was obtained from an endophytic fungus A. porosus [87] and recently gained interest as a cytotoxic agent, showing selectivity toward melanoma and kidney cancer cell lines with a unique mechanism of action targeting regulatory volume increase [136]. Arugosin C (291) was a novel prenylated polyketide produced by a marine-derived fungus, A. versicolor HBU-2017-7, but exhibited no inhibitory activity against HCV protease [119,137]. Chlovalicin (292) was determined as a new chlorinated meroterpenoid from strain A. niger BRF-074 and displayed no cytotoxicity towards the HCT-116 cell line [138].

Xanthones
Xanthones are a class of natural products with hetero-tricyclic structures possessing a variety of biological activities, including antihypertensive, anticonvulsant, antithrombotic, an- Additionally, chemical analysis of the liquid cultures of strain TJ23 resulted in the discovery of two novel terpene-polyketide hybrids (287 and 288), of which compound 287 was a potential inhibitor of PBP2a and worked synergistically with the β-lactam antibiotics oxacillin and piperacillin against MRSA [135]. Sphaeropsidin A (289), along with aspergiloid E (290), was obtained from an endophytic fungus A. porosus [87] and recently gained interest as a cytotoxic agent, showing selectivity toward melanoma and kidney cancer cell lines with a unique mechanism of action targeting regulatory volume increase [136]. Arugosin C (291) was a novel prenylated polyketide produced by a marinederived fungus, A. versicolor HBU-2017-7, but exhibited no inhibitory activity against HCV protease [119,137]. Chlovalicin (292) was determined as a new chlorinated meroterpenoid from strain A. niger BRF-074 and displayed no cytotoxicity towards the HCT-116 cell line [138].

Xanthones
Xanthones are a class of natural products with hetero-tricyclic structures possessing a variety of biological activities, including antihypertensive, anticonvulsant, antithrombotic, antitumor and so on [139][140][141][142][143]. Two new xanthones (293 and 294, Figure 19) were purified from a marine sponge-derived fungus A. versicolor [85], and compound 293, along with its derivative (295), was also obtained from strain A. versicolor HBU-2017-7 and shown to have significant cytotoxicity [119]. By continuous cultivation for activating silent polyketide BGCs in strain A. nidulans FGSCA4, a new prenylated cytotoxic xanthone (296) was discovered in its chemostat cultures [144]. Two xanthone dimers (297 and 298) originally produced by A. aculeatus in 1977 were rediscovered from strains A. sp. SCSIO XWS03F03 and A. aculeatus IBT 21030 [65,145]. Bioassay-guided fractionation of the crude extract of a soil fungus A. terreus X3 resulted in the isolation of penicitrinones A and B (299 and 300), which the former showed moderate activity against B. megaterium with a MIC value of 1.60 µM [29]. Four prenylated xanthones (301-304) were separated from the rice medium of the endophytic strain A. sp. TJ23 exhibited weak inhibitory activities against the growth of B16, HepG2, and LLC cancer cell lines [58]. Chemical analysis of a marine sponge-derived strain A. europaeus WZXY-SX-4-1 afforded six xanthone polyketides (305-310), of which compounds 305 and 310 exerted excellent down-regulation of NF-κB in LPS-induced SW480 cells [16]. Oxisterigmatocystin I (311), along with four analogs (293, 312-314), were purified from the culture of a sponge-derived strain A. sp. F40 and showed weak antimicrobial activity against S. aureus [90]. When cultured under static conditions, strain A. sydowii SCSIO 41301 was found to produce two new xanthones (315 and 316), which exhibited obvious selective inhibitory activity against H1N1 influenza [33].

Miscellaneous
A number of other bioactive polyketides had been discovered and identified from Aspergillus strains. Mevinolin (317, Figure 20), along with its analog 318, was obtained from strain A. terreus ATCC 20542 and exhibited a potent competitive inhibitory effect on hydroxymethylglutaryl coenzyme A (HMG CoA) reductase [146]. Aspermytin A (319) was a new neurotrophic agent produced by a mussel-derived strain of Aspergillus [147]. Three decaline derivatives (320-322) showed significant cytotoxicity against melanoma cell lines [148,149]. Calbistrin A (323), together with its analog (324) derived from strain A. aculeatus IBT 21030, acted as an excellent antifungal agent, a promoter of nerve growth factor (NGF) production and a cholesterol-lowering substance [150,151]. Two lovastatin analogs (325 and 326) were detected in the solid culture of A. versicolor SC0156 [152]. Aspergones N-Q (327-330), along with epoxyquinol (331) were separated from the fermentation broth of A. sp. OUCMDZ-1583 and displayed strong α-glucosidase inhibitory effects [45]. Salimyxin B (332) produced by the endophytic strain A. sp. TJ23 showed inhibitory activities against HepG2 with an IC 50 value of 9.87 µM [58]. Hexylitaconic acid (333) was a binary fatty acid originally derived from a marine-derived fungus Arthrinium sp., was also produced by the strain of A. niger and showed potent antibacterial and antioxidant activities as well as good inhibitory effect on acetylcholinesterase and p53-HDM2 interaction [41,153,154].  A terrein glucoside (334) was a new angiogenin secretion inhibitor produced by strain A. sp. PF1381 [155]. Bioassay-guided isolation of an extract of A. sp. MF6215 led to the discovery of three novel 11-membered macrocyclic biphenyl ether lactones (335-337), in which compound 335 inhibited the IgE binding to its receptor by an IC 50 value of 200 µM [156]. By UHPLC-DAD-HRMS and dereplication, aculenes C and D (338 and 339) were isolated from a strain of A. aculeatus but showed weak antifungal activity [150]. Dehydrocurvularin (340) was a new lactone polyketide from strain A. terreus ATCC 20542 and acted as a prevalent fungal phytotoxin with heat shock response and immune-modulatory activities and a broad-spectrum inhibitor of various cancer cell lines in vitro [61,157,158]. Aspergones G and H (341 and 342) produced by the strain A. sp. OUCMDZ-1583 displayed no cytotoxic activity [45]. A. flavus-derived terrein (343) was a novel suppressor of ABCG2-expressing breast cancer cells MCF-7 cells [13,30].

Conclusions and Perspectives
In summary, the genus Aspergillus is a prolific source of polyketides with diverse chemical structures and a variety of biological activities. Many of these substances or derivatives have therapeutic effects, such as the immunosuppressant agent (3), the antioxidant benzaldehydes (34,35), the α-glucosidase inhibitors (327-330), etc. Furthermore, the potential to discover novel polyketides from Aspergillus strains is still immense since a great number of their BGCs are shown to be inactive or unawakened under traditional culture conditions [159]. With the development and application of bioinformative tools and analytical techniques, more and more Aspergillus genomes, as well as functional genes, will be sequenced and annotated. These silent BGCs responsible for the biosynthesis of novel polyketides are being disclosed and activated using new strategies, such as the one strain many compounds (OSMAC) approach and genome mining combined with metabolic engineering [8,160,161]. In addition, the biosynthesis of polyketides from acyl-CoA thioesters is catalyzed by various PKSs, which structures of initiation and condensation domains provide valuable insights into the molecular factors governing starter unit selectivity and chain-length control. A detailed understanding of these PKS structural features controlling polyketide biosynthesis and modification offers a powerful tool for the controlled and rational design of novel polyketides through enzyme engineering. Therefore, more efforts should be made to employ biosynthetic engineering approaches to improve the efficient discovery of novel polyketides from the genus Aspergillus.